NORTHERN CARIBBEAN UNIVERSITY

College of Natural and Applied Sciences, Allied Health and Nursing

Department of Medical Technology
BCHM 501: General Biochemistry

Worksheet 4 – Overview of Enzyme Kinetics

1. BRIEFLY discuss the general effect of the following on the rate of enzyme catalysed
reactions: [10 pts]
a. pH

Enzymes are active only within a limited range of pH,. But the limits may be wide, e.g. pH 5 to 10, or
narrow, e.g. over 1 pH unit. Within the range there will be an optimum at which the maximum activity
(the highest value for Vmax) is attained hence increased rate: this could be a short range in itself. If the
pH is not optimum this could have a decrease in rate. This is likely because pH affects ionic and
hydrogen bonds which are important to enzyme shape and therefore enzyme activity (Reece, et al
2010). The effect of pH is generally tested at high substrate concentration. It should be noted that the
optimum pH is reaction direction-specific. (Scopes.R, 1993)

b. Temperature
Temperature affects enzyme activity in much the same way as it affects other chemical reactions by
increasing the kinetic energy of the system/ collissions. Rates increase by between 4 and 8% per
degree C, although at high temperatures denaturation of the enzyme protein decreases product
formation. Thus it is important when carrying out an enzyme assay to ensure that the temperature
remains constant, and also that you know exactly what it is. For comparison with other results that
may have been reported at other temperatures, the exact temperature coefficient should be known; if
not, a figure of 6% per degree is a useful approximation.

c. Substrate concentration
The traditional enzyme has a hyperbolic response to substrate concentration, according to the
Michaelis– Menten equation. From this equation we understand that the rate measured varies with
substrate concentration more rapidly as the substrate concentration decreases. In essence enzymes will
work best if there is plenty of substrate as increased collisions will be possible. As the concentration of
the substrate increases, so does the rate of enzyme activity. However, the rate of enzyme activity does
not increase forever. This is because a point will be reached when the enzymes become saturated and
no more substrates can fit at any one time even though there is plenty of substrate available. There are
many enzymes which do not obey the simple Michaelis–Menten formula. The most extreme
deviations are with those enzymes known as ‘allosteric’, in which a sigmoid shape of response is
found.
d. Enzyme concentration
As long as there is substrate available to bind to, increasing enzyme concentration will speed up the
enzymatic reaction. Once all of the substrate is bound, the reaction will no longer speed up with the
increasing enzyme concentration, since there will be nothing for additional enzymes to bind to. Often a
small amount of enzyme can consume a large amount of substrate. However, with the increase of
enzyme concentration, the effectiveness of the active sites also increases, so these active sites will
convert the substrate molecules into products. This basically means that if the concentration of the
enzyme is to be increased, there needs to be an excess of substrate, which means that the reaction must
be independent of the concentration of the substrate.

e. The presence of an inhibitor

There are different types of inhibitors for example:
Non-competitive inhibitors:

The induced-fit theory explains a number of anomalous properties of enzymes. An example is

“noncompetitive inhibition,” in which a compound inhibits the reaction of an enzyme but does not
prevent the binding of the substrate. In this case, the inhibitor compound attracts the binding group so
that the catalytic group is too far away from the substrate to react. The site at which the inhibitor binds
to the enzyme is not the active site and is called an allosteric site. The inhibitor changes the shape of
the active site to prevent catalysis without preventing binding of the substrate. An inhibitor also can
distort the active site by affecting the essential binding group; as a result, the enzyme can no longer
attract the substrate and hence rate decreases.
Competitive inhibitors:

Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an
enzyme. In that case, almost all the active sites of almost all the enzyme molecules will be occupied by
the competitive inhibitor instead of substrate and hence rate decreases.
Suicide inhibitor
In contrast to the previous two types of inhibitors, which involve reversible binding of the inhibitor to
the enzyme, suicide inhibition is irreversible because the inhibitor becomes covalently bound to the
enzyme during the inhibition and thus cannot be removed. Suicide inhibition rather closely resembles
competitive inhibition because the inhibitor generally resembles the substrate and binds to the active
site of the enzyme and hence rate decreases.

2. What is the specific purpose of the Michaelis-Menten equation? [2 pts].

The specific purpose of the Michaelis-Menten equation lies in the fact that we can use the
Michaelis-Menten equation to describe the reaction rate of an enzyme catalysed reaction
thereby enabling a better understanding of how enzymes affect chemical reactions. It shows
that a traditional enzyme has a hyperbolic response to substrate concentration.
 3. Use the Michaelis-Menten equation to explain why the rate of an enzyme-catalysed reaction is
proportional to the amount of enzyme. [4 pts]

The Michaelis-Menten equation:

V ×[ S]
ʋ = Kmaxm+ [ S ]
Where [S] is the substrate concentration, v is the rate measured, Vmax is the maximum theoretical rate
at infinite substrate concentration, and Km is the Michaelis- Menten constant.
However further extrapolation of this equation can be done due to the relationship of Vmax to the
catalytic constant of given enzyme( kcat ) and enzyme conentration [ E ] .

V max × [ S ] K cat × [ E ] × [ S ]
ʋ= K m+ [ S ] = Km+ [ S ]

This shows that indeed the rate of a enzyme catalyzed reaction is directly proportional to the Enzyme
concentration as per its position in the equation as a numerator. Moreover from a biochemical
perspective this is correct based on the fact that if the concentration of the enzymes increases this
auomatically allows for the increased presence of enzyme active sites that can be occupied with
substrates thereby increasing the reaction rate ʋ of product formation.

4. Use the Michaelis-Menten equation to explain why the rate of an enzyme-catalysed reaction
reaches a maximum value at high substrate concentration. [4 pts]

V max × [ S ] K ×[ E ] ×[ S ]
ʋ= K m+ [ S ]
=¿ cat
Km+ [ S ]

Acoording to the Michaelis-Menten equation above the rate of an enzyme-catalysed reaction

reaches a maximum value V max at high substrate concentration. This is true for Vmax is
directly proportional to Enzyme concentration, the higher [ E ] the higher Vmax will be. It is at
the point Vmax where all of the available enzyme active sites would be occupied with
substrates. Therefore, increasing the substrate concentration further will not change the rate
of product formation for even though at the beginning when the substrate concentration was
increased the reaction rate increased it will eventually asymptotically approach the saturation
rate Vmax as a lack of active sites becomes present due to the saturation of the Enzyme active
sites with substrates.

5. Using graphical sketches, explain the difference(s) in effect of competitive and non-
competitive inhibitors on enzyme catalysis. [8 pts]

Competitive and non-competitive inhibitors can be told

apart by how they affect an enzyme's activity at different
substrate concentrations.
On a graph of reaction velocity (y-axis) vs substrate concentrations (x-axis), you can differentiate the
two types of inhibitors by the shape of the curves:
If an inhibitor is competitive, it will decrease reaction rate when there's not much substrate, but can be
"out-competed" by lots of substrate/ increasing substrate concentration. That is, the enzyme can still
reach its maximum reaction rate ( Vmax) given enough substrate. In that case, almost all the active
sites of almost all the enzyme molecules will be occupied by the substrate rather than the inhibitor. 
However if an inhibitor is non-competitive, the enzyme-catalysed reaction will never reach its normal
maximum rate( Vmax) even with a lot of substrate. This is because the shape active sites of the
enzyme molecules with the non-competitive inhibitor bound are altered and will not accept allow an
induced fit to occur regardless of how much substrate is available. (Tymoczsko. L, 2002)
6. Arsenic is widely known to be poisonous to humans due its role in enzyme inhibition. Discuss
the effect of arsenic on the citric acid cycle and explain the type of inhibition (competitive or
non-competitive) involved. [10 pts]

The enzyme complex pyruvate dehydrogenase (PDH) oxidizes pyruvate to acetyl-CoA, a precursor to
intermediates of the citric acid cycle. The citric acid cycle degrades the intermediates, providing the
mitochondria with reducing equivalents needed for electron transport and the subsequent production of
ATP.
Arsenic exists in two oxidation states—arsenate, As(III), and arsenite, As(V)—both of which are
anions. Arsenite is more lethal than arsenate and is an oxyanion of trivalent arsenic AsO33- (or
protonated derivatives). Arsenite interferes with cellular longevity by  inhibition of an essential
metabolic enzyme pyruvate dehydrogenase (PDH) complex ( It can also do the same to α-ketoglutarate
dehydrogenase complex in the TCA cycle by inhibiting it), which catalyses the oxidation
of pyruvate to acetyl-CoA with the aid of the cofactor CoA and also NAD+. Inhibition of the PDH
complex can block the citric acid cycle and ultimately decrease the production of ATP, resulting in
cell damage and death as the energy system of the cell is disrupted resulting in a
cellular apoptosis episode.
Mechanism of Arsenite inhibiting of pyruvate dehydrogenase
The mechanism employed by
arsenite involves it acting as a
molecular analogue of
phosphate (HPO42-)
Therefore it can compete for
phosphate anion transporters
and replace phosphate in
biochemical reactions when
for example the phosphate
group of CoA tries to bind to
the thiol groups of pyruvate
dehydrogenase complex or α-ketoglutarate dehydrogenase complex. (Hughes F, 2002). Hence the type
of regulation that takes place is competitive inhibition as the arsenite inhibitor is an analog of
phosohate and generally resembles it and binds to the active site of the enzyme. For example using the
diagram, the generation of adenosine-5′-triphosphate (ATP) during oxidative phosphorylation can be
inhibited by the replacement of phosphate with the arsenite radical. This occurs as trivalent arsenicals
have high affinity for sulfhydryl groups (Thiol groups) of the lipoic acid moiety (Dihydrolipoamide)
of the PDH complex, component E3, resulting in a reduced conversion of pyruvate to acetyl
coenzyme-A. This is due to the arsenic being chelated onto the enzyme Pyruvate dehydrogenase
complex so that both the citric acid cycle activity and production of cellular ATP are decreased.
(Islam. K, 2015). An antidote that can counteract arsenic poisoning was developed by the British in the
first world war and was called 2,3 -Dimercaptopropanol,(British anti-lewisite, BAL), which could bind
arsenic to form very stable complexes, preventing arsenic from binding to pyruvate dehydrogenase
complex or α-ketoglutarate dehydrogenase complex. (Shen. S, 2013). The stable complexes formed
have polar regions that allows it to be excreted from the body easily.

References
1.  Hughes M. F. Toxicol. 2002, PubMed, Google Scholar.com
2. Khairul Islam, Hua Naranmandura, Advances in Molecular Toxicology, 2015
3. Khan, K. (2020). Enzyme regulation (article). Khan Academy.
https://www.khanacademy.org/science/ap-biology/cellular-energetics/environmental-impacts-
on-enzyme-function/a/enzyme-regulation#:%7E:text=The%20competitive%20inhibitor
%20binds%20to,longer%20catalyze%20the%20reaction%20efficiently.
4. Reece, et al, Campells Reece Biology 11th ed. 2016, Pearson Publishing.
5. Scopes. R, Encyclopedia of life sciences, 2002 Macmillan Publishers Ltd, Nature Publishing
Group
6. Tymoczko,L, et al. Biochemistry, 5th edition, 2002. NewYork